The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD⁺ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD⁺ turnover, which makes NAD⁺ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD⁺ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD⁺ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.The cyanoguanidinopyridine GMX1778 (previously known as CHS828) is the active form of the prodrug GMX1777 and has potent antitumor activity in vitro and in vivo against cell lines derived from several different tumor origins (11). The antitumor activity of GMX1778 has been widely studied since its discovery (1, 11, 19-21, 24), but positive identification of the molecular target and the mechanism of action of GMX1778 has been elusive. Here, we demonstrate that GMX1778 exerts its antitumor activity via its potent and selective antagonism of NAD⁺ biosynthesis. GMX1777 is currently being assessed in phase I clinical trials for treatment of patients with refractory solid tumors.The pyridine nucleotide NAD⁺ plays a major role in the regulation of several essential cellular processes (7, 22, 25, 38). In addition to being a biochemical cofactor for enzymatic redox reactions involved in cellular metabolism, including ATP production, NAD⁺ is important in diverse cellular pathways responsible for calcium homeostasis (17), gene regulation (5), longevity (18), genomic integrity (33), and apoptosis (36). Cancer cells exhibit a significant dependence on NAD⁺ for support of the high levels of ATP production necessary for rapid cell proliferation. They also consume large amounts of this cofactor via reactions that utilize poly(ADP) ribosylation, including DNA repair pathways (10, 37, 39).In eukaryotes, the biosynthesis of NAD⁺ occurs via two biochemical pathways: the de novo pathway, in which NAD⁺ synthesis occurs through the metabolism of l-tryptophan via the kynurenine pathway, and the salvage pathway. The NAD⁺ salvage pathway can use either nicotinamide (niacinamide) (NM) or nicotinic acid (niacin) (NA) (via the Preiss-Handler pathway) as a substrate for NAD⁺ production. Saccharomyces cerevisiae species predominantly use NA as the substrate for NAD⁺ biosynthesis, through the deamidation of NM by the nicotinamidase PNC1 (25). However, mammalian cells do not express a nicotinamidase enzyme and use NM as the preferred substrate for the NAD⁺ salvage pathway. The mammalian NAD⁺ biosynthesis salvage pathway using NM is composed of NA phosphoribosyltransferase (NAMPT), which is the rate-limiting and penultimate enzyme that catalyzes the phosphoribosylation of NM to produce nicotinamide mononucleotide (NMN) (27, 29). NMN is subsequently converted to NAD⁺ by NMN adenyltransferases (NMNAT). The gene encoding NAMPT was originally identified as encoding a cytokine named pre-B-cell colony-enhancing factor (PBEF1) (30). NAMPT was also identified as a proposed circulating adipokine named visfatin (thought to be secreted by fat cells) and was suggested to function as an insulin mimetic; however, this role of NAMPT currently remains controversial (8). In mice, NAMPT has been shown to act as a systemic NAD⁺ biosynthetic enzyme that regulates insulin secretion from β cells (28). The molecular structure of NAMPT from human (15), rat (16) and mouse (35) tissue, containing either NMN or the inhibitor APO866, have been determined by X-ray crystallography. These structures revealed that NAMPT is a dimeric type II phosphoribosyltransferase.Here, we report that the anticancer compound GMX1778 is a specific inhibitor of NAMPT in vivo and in vitro and is itself a substrate for the enzyme. Phosphoribosylated GMX1778 inhibits NAMPT as potently as GMX1778 but is preferentially retained within cells. Finally, we have identified a novel anticancer strategy utilizing NA rescue of GMX1778 cytotoxicity to increase the therapeutic index of GMX1777 activity in tumors that are deficient in NA phosphoribosyltransferase 1 (NAPRT1).     

The KtrA and KtrE Subunits Are Required for Na+-Dependent K+ Uptake by KtrB across the Plasma Membrane in Synechocystis sp. Strain PCC 6803

The Na⁺-dependent K⁺ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K⁺-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K⁺ uptake and in modulating Na⁺ dependency. Expression of KtrB alone in a K⁺ uptake-deficient Escherichia coli strain conferred low K⁺ uptake activity that was not stimulated by Na⁺. Coexpression of both KtrA and KtrE with KtrB increased the K⁺ transport activity in a Na⁺-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent K_m for K⁺ in all cases. However, none of the mutations eliminated the Na⁺ dependency of Ktr-mediated K⁺ transport. Mutations of residues on the cytoplasmic side had larger effects on K⁺ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K⁺ uptake rate and conferred Na⁺ dependency.Cyanobacterium Synechocystis sp. strain PCC 6803 contains a number of different K⁺ uptake systems that may contribute to satisfying its requirement of K⁺ (3, 19, 36). Among these systems, Ktr has been shown to have a major role not only in K⁺ uptake but also in adaptation against high-osmolarity stress (3, 19). Inactivation of the ktr gene renders the cells hypersensitive to high concentrations of NaCl and the nonionic compound sorbitol. Ktr-mediated K⁺ uptake depends on the presence of Na⁺ in the medium, which is likely to be an adaptation to salinity stress. A requirement of Na⁺ for K⁺ transport activity has also been found in the homologous protein from Vibrio alginolyticus (21). This dependency on Na⁺ is a unique property of Ktr-type transporters and has not been found in other types of K⁺ transporters or channels (32). The structure and function of Ktr-type transporters have been studied in a number of organisms (3, 6, 7, 9, 11-14, 18-20, 30, 32-34). The Ktr system from Synechocystis consists of three subunits, KtrA, KtrB, and KtrE (19). The KtrE gene and the KtrB gene form a cistron, whereas the KtrA gene resides at a site distant from the KtrEB genes in the Synechocystis genome (19). KtrB, the K⁺-translocating subunit, is a member of the Ktr/Trk/HKT family of K⁺ transporters. These transporters have been proposed to have evolved from two membrane-spanning K⁺ channels (6, 7). According to the model, this type of transporter contains eight transmembrane domains, which consist of a 4-fold-repeated membrane-pore-membrane (M1-P-M2) motif (6, 7, 13, 18). An intramolecular electrostatic interaction of Synechocystis KtrB has been proposed to stabilize the protein in its active configuration (12). In addition, a conserved His in the external region in Synechocystis KtrB has been shown to be crucial for KtrB function (39). The region of the Vibrio Ktr protein responsible for gating of ion permeation has been identified (9). However, not much is known about the mechanism of Na⁺ binding to KtrB in Synechocystis.The KtrA subunit belongs to the family of KTR (K⁺-transport nucleotide binding)/RCK (regulating the conductance of K⁺ channels) proteins, which contain a Rossmann-fold sequence encoding β-α protein structure for NAD⁺/NADH binding (17). Accordingly KtrA has been proposed to regulate the K⁺ transport activity of KtrB by changing its binding from NAD⁺ to NADH through a ligand-mediated conformational switch mechanism (25). It has also been shown that ATP promotes complex formation between KtrA and KtrB and that KtrAB from V. alginolyticus when expressed in Escherichia coli cells requires both ATP and the membrane potential for its activity (17).KtrE is a unique subunit found only in Synechocystis; it is not involved in KtrB-mediated K⁺ transport in V. alginolyticus and Bacillus subtilis (11, 32). The termination codon of ktrE overlaps the initiation codon of ktrB in the same cistron, which has not been found in other bacterial ktrB-related genes. Coexpression of KtrA with KtrB alone does not complement the growth defect of an E. coli K⁺ uptake mutant. However, introduction of KtrE into the same mutant background in addition to KtrA and KtrB complements the mutation of the K⁺ uptake system (19). Interestingly, the KtrE protein has been shown to function as a digalactosyldiacylglycerol (DGDG) synthase (EC 2.4.6.241), an enzyme that produces DGDG from monogalactosyldiacylglycerol (MGDG). KtrE has therefore also been designated DgdA (1). Under nonstress conditions, DGDG is found in the thylakoid membranes, which helps stabilize the photosystem II complex in Synechocystis (29). Under phosphate-limited conditions, DGDG is synthesized instead of phospholipids in Synechocystis (1). However, KtrB functions as a major K⁺-conducting transport pore in the Synechocystis plasma membrane. The subcellular localization of KtrE has not been identified directly. Inactivation of ktrE (also called dgdA) in Synechocystis does not result in sensitivity to osmotic stress imposed by 300 mM sorbitol (1). This may be inconsistent with the requirement of KtrE for KtrB-mediated K⁺ uptake in the presence of KtrA in the E. coli expression system (19).Because of these uncertainties about the roles of the KtrA and KtrE subunits in K⁺ uptake by KtrB in Synechocystis and about the identity of the Na⁺ binding site in KtrB, we examined the subcellular localization and membrane association of KtrA and KtrE, the requirement of these subunits for KtrB-mediated K⁺ uptake, and the primary target for Na⁺ binding in KtrB.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Simian Immunodeficiency Virus-Specific CD8+ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection

The kinetics of CD8⁺ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8⁺ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4⁺ T cells early after SIV infection.The antiviral activity of AIDS virus-specific CD8⁺ T cells is well documented in both in vivo (1, 4, 21) and in vitro (8, 24, 29) studies. Accordingly, human immunodeficiency virus (HIV) vaccine modalities that focus on engendering antiviral CD8⁺ T cells are being developed (13, 26, 28). Ideally, a CD8⁺ T cell-based vaccine would stimulate responses against epitopes that are presented by major histocompatibility complex class I (MHC-I) molecules early after infection of a target cell. However, successful selection of antigenic sequences for a CD8⁺ T cell-based vaccine has been frustrated in part by an incomplete understanding of the properties of effective CD8⁺ T cell responses (25).     

Effective Simian Immunodeficiency Virus-Specific CD8+ T Cells Lack an Easily Detectable,Shared Characteristic

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8⁺ T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8⁺ T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8⁺ T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8⁺ T cells to suppress viral replication from SIVmac239-infected CD4⁺ T cells. Using this assay, we established an antiviral hierarchy when we compared CD8⁺ T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8⁺ T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8⁺ T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8⁺ T-cell response may be comprised of several factors.CD8⁺ T cells may play a critical role in blunting peak viremia and controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. The transient depletion of CD8⁺ cells in SIV-infected macaques results in increased viral replication (26, 31, 51, 70). The emergence of virus-specific CD8⁺ T cells coincides with the reduction of peak viremia (12, 39, 42, 63), and CD8⁺ T-cell pressure selects for escape mutants (6, 9, 13, 28, 29, 38, 60, 61, 85). Furthermore, particular major histocompatibility complex (MHC) class I alleles are overrepresented in SIV- and HIV-infected elite controllers (15, 29, 33, 34, 46, 56, 88).Because it has been difficult to induce broadly neutralizing antibodies (Abs), the AIDS vaccine field is currently focused on developing a vaccine designed to elicit HIV-specific CD8⁺ T cells (8, 52, 53, 82). Investigators have tried to define the immune correlates of HIV control. Neither the magnitude nor the breadth of epitopes recognized by virus-specific CD8⁺ T-cell responses correlates with the control of viral replication (1). The quality of the immune response may, however, contribute to the antiviral efficacy of the effector cells. It has been suggested that the number of cytokines that virus-specific CD8⁺ T cells secrete may correlate with viral control, since HIV-infected nonprogressors appear to maintain CD8⁺ T cells that secrete several cytokines, compared to HIV-infected progressors (11, 27). An increased amount of perforin secretion may also be related to the proliferation of HIV-specific CD8⁺ T cells in HIV-infected nonprogressors (55). While those studies offer insight into the different immune systems of progressors and nonprogressors, they did not address the mechanism of viral control. Previously, we found no association between the ability of SIV-specific CD8⁺ T-cell clones to suppress viral replication in vitro and their ability to secrete gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-2 (IL-2) (18).Evidence suggests that some HIV/SIV proteins may be better vaccine targets than others. CD8⁺ T cells recognize epitopes derived from Gag as early as 2 h postinfection, whereas CD8⁺ T cells specific for epitopes in Env recognize infected cells only at 18 h postinfection (68). Additionally, a previously reported study of HIV-infected individuals showed that an increased breadth of Gag-specific responses was associated with lower viral loads (35, 59, 65, 66). CD8⁺ T-cell responses specific for Env, Rev, Tat, Vif, Vpr, Vpu, and Nef were associated with higher viral loads, with increased breadth of Env in particular being significantly associated with a higher chronic-phase viral set point.None of the many sophisticated methods employed for analyzing the characteristics of HIV- or SIV-specific immune responses clearly demarcate the critical qualities of an effective antiviral response. In an attempt to address these questions, we developed a new assay to measure the antiviral efficacy of individual SIV-specific CD8⁺ T-cell responses sorted directly from fresh peripheral blood mononuclear cells (PBMC). Using MHC class I tetramers specific for the epitope of interest, we sorted freshly isolated virus-specific CD8⁺ T cells and determined their ability to suppress virus production from SIV-infected CD4⁺ T cells. We then looked for a common characteristic of efficacious epitope-specific CD8⁺ T cells using traditional methods.     

Sti1 Regulation of Hsp70 and Hsp90 Is Critical for Curing of Saccharomyces cerevisiae [PSI+] Prions by Hsp104

Although propagation of Saccharomyces cerevisiae prions requires Hsp104 protein disaggregating activity, overproducing Hsp104 “cures” cells of [PSI⁺] prions. Earlier evidence suggests that the Hsp70 mutant Ssa1-21 impairs [PSI⁺] by a related mechanism. Here, we confirm this link by finding that deletion of STI1 both suppresses Ssa1-21 impairment of [PSI⁺] and blocks Hsp104 curing of [PSI⁺]. Hsp104''s tetratricopeptide repeat (TPR) interaction motif was dispensable for curing; however, cells expressing Sti1 defective in Hsp70 or Hsp90 interaction cured less efficiently, and the Hsp90 inhibitor radicicol abolished curing, implying that Sti1 acts in curing through Hsp70 and Hsp90 interactions. Accordingly, strains lacking constitutive or inducible Hsp90 isoforms cured at reduced rates. We confirm an earlier finding that elevating free ubiquitin levels enhances curing, but it did not overcome inhibition of curing caused by Hsp90 defects, suggesting that Hsp90 machinery is important for the contribution of ubiquitin to curing. We also find curing associated with cell division. Our findings point to crucial roles of Hsp70, Sti1, and Hsp90 for efficient curing by overexpressed Hsp104 and provide evidence supporting the earlier suggestion that destruction of prions by protein disaggregation does not adequately explain the curing.Saccharomyces cerevisiae prions are self-replicating misfolded forms of normal cellular proteins. They are believed to propagate as amyloid, which is a highly ordered fibrous aggregate. What triggers prion formation is uncertain, but in order to be maintained in an expanding yeast population, prions must grow, replicate, and be transmitted to daughter cells during cell division. Growth occurs when soluble protein joins the fiber ends and is converted into the prion form (30, 52, 58). Replication is associated with fragmentation of prion polymers, which generates new prions from preexisting material (37, 50). Transmission is believed to occur by passive diffusion of prions with cytoplasm (57).Although it is uncertain to what extent cellular factors influence growth or transmission of prions, it is clear that the Hsp104 disaggregation machinery is necessary for prion replication (10, 17, 55, 70). Hsp104 is a hexameric AAA+ chaperone that protects cells from a variety of stresses by resolubilizing proteins from aggregates (24, 25, 53). With help from Hsp70 and Hsp40, it extracts monomers from aggregates and extrudes them through its central pore (24, 41, 68). This machinery could act in prion replication by extracting monomers from amyloid fibers (29, 68), which would destabilize the fibers, causing them to break into more numerous pieces that each can continue to propagate the prion.Paradoxically, overexpressing Hsp104 very efficiently “cures” cells of the [PSI⁺] prion, which is composed of the translation termination factor Sup35 (10). A widely held view of this curing is that elevating the cellular protein disaggregation activity causes complete destruction of prions. However, elevating Hsp104 has little or no effect on most other amyloidogenic prions (15, 16, 38, 47, 54, 66), although it can be inferred to cure [MCA] prions in cells also propagating a prion of an Mca1-Sup35 fusion (49). Together, these results suggest that prions of Sup35, and perhaps those of Mca1, are particularly sensitive to Hsp104 disaggregation activity. Alternatively, something in addition to or other than a simple increase in protein disaggregation is involved in the curing.Although protein disaggregation activity of Hsp104 is required for both thermotolerance and prion propagation, we and others have identified mutations in Hsp104 that affect these processes separately (27, 32, 39, 60). The ability of Hsp104 to thread proteins through its central pore, however, is required for both processes (29, 41, 68), so this distinction in Hsp104 function could be due to differences in how Hsp104 interacts with amorphous aggregates of thermally denatured proteins and highly ordered prion aggregates or with cofactors that interact with the different prions as substrates. In any scenario, efficiency and specificity of Hsp104 function are affected by interactions with other components of the disaggregation machinery, in particular the Hsp70s and Hsp40s, which are believed to interact first with substrates to facilitate action of Hsp100 family disaggregases (2, 71, 72).Increasing expression of either ubiquitin (Ub) or Ssb, an Hsp70 that has roles in protein translation and proteasome degradation, enhances Hsp104 curing of [PSI⁺] (3, 11, 12). Predictably, reducing expression of either of them reduces curing efficiency. The mechanisms underlying these effects are unknown, but the combined effects of Ssb and Ub are additive, suggesting that they act in different pathways. The role of Ub is indirect, as Sup35 is neither ubiquitylated nor degraded during curing. Whether other chaperones are involved in the effects of Ub on curing has not been investigated.Earlier we isolated a mutant of the Hsp70 Ssa1, designated Ssa1-21, that weakens and destabilizes [PSI⁺] propagation (33). We later isolated several Hsp104 mutants that suppress this antiprion effect (29). The Hsp104 mutants retain normal functions in thermotolerance, protein disaggregation, and prion propagation, but when overexpressed, they are unable to cure [PSI⁺], even in wild-type cells. These findings argue against a specific hypersensitivity of [PSI⁺] to disaggregation and support the notion that something distinct from or in addition to complete destruction of prions is involved in the curing. They also imply that Ssa1-21 and elevated Hsp104 inhibit [PSI⁺] prions by similar mechanisms. A prediction from this conclusion is that other suppressors of Ssa1-21 will also inhibit curing of [PSI⁺] by overexpressed Hsp104. Indeed, we find here that alterations that suppress Ssa1-21 inhibition of [PSI⁺] do interfere with curing of [PSI⁺] by overexpressed Hsp104. We also provide evidence that Hsp90 has a critical role in this curing and that the ability of Ub to enhance curing depends on proper function of Hsp90 machinery.     

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from,but Overlaps with,the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4⁺ and CD8⁺ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4⁺ T cells, and HTLV-2 SU, which primarily binds to activated CD8⁺ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4⁺ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.The primate T-cell lymphotropic virus (PTLV) group of deltaretroviruses consists of three types of human T-cell lymphotropic viruses (HTLVs) (HTLV-1, HTLV-2, HTLV-3), their closely related simian T-cell lymphotropic viruses (STLVs) (STLV-1, STLV-2, STLV-3), an HTLV (HTLV-4) for which a simian counterpart has not been yet identified, and an STLV (STLV-5) originally described as a divergent STLV-1 (5-7, 30, 35, 37, 38, 45, 51, 53). HTLV-1 and HTLV-2, which have a 70% nucleotide homology, differ in both their pathobiology and tropism (reviewed in reference 13). While HTLV-1 causes a neurological disorder (tropical spastic paraparesis/HTLV-1-associated myelopathy) and a hematological disease (adult T-cell leukemia/lymphoma) (15, 42, 55), HTLV-2 is only rarely associated with tropical spastic paraparesis/HTLV-1-associated myelopathy-like disease and is not definitively linked to any lymphoproliferative disease (12, 20). In vivo, both HTLV-1 and HTLV-2 infect T cells. Although HTLV-1 is primarily found in CD4⁺ T cells, other cell types in the peripheral blood of infected individuals have been found to contain HTLV-1, including CD8⁺ T cells, dendritic cells, and B cells (19, 29, 33, 36, 46).Binding and entry of retroviruses requires specific interactions between the Env glycoproteins on the virus and cell surface receptor complexes on target cells. For HTLV-1, three molecules have been identified as important for entry, as follows: heparan sulfate proteoglycans (HSPGs), neuropilin-1 (NRP-1), and glucose transporter 1 (GLUT-1) (16, 22, 26, 28, 29, 34, 39, 44). Recent studies support a model in which HSPG and NRP-1 function during the initial binding of HTLV-1 to target cells, and GLUT-1 functions at a postattachment stage, most likely to facilitate fusion (29, 34, 49). Efficient HTLV-2 binding and entry requires NRP-1 and GLUT-1 but not HSPGs (16, 26, 39, 49).This difference in the molecules required for binding to target cells reflects differences in the T-cell tropisms of these two viruses. Activated CD4⁺ T cells express much higher levels of HSPGs than CD8⁺ T cells (26). In infected individuals, HTLV-1 is primarily found in CD4⁺ T cells, while HTLV-2 is primarily found in CD8⁺ T cells (21, 43, 46). In vitro, HTLV-1 preferentially transforms CD4⁺ T cells while HTLV-2 preferentially transforms CD8⁺ T cells, and this difference has been mapped to the Env proteins (54).We and others have reported the discovery of HTLV-3 in two Cameroonese inhabitants (6, 7, 53). We recently uncovered the presence of a third HTLV-3 strain in a different population living several hundred kilometers away from the previously identified groups (5), suggesting that this virus may be common in central Africa. Since the HTLV-3 sequences were obtained by PCR amplification of DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals, little is known about its tropism and pathobiology in vivo. Based on the correlation between HSPG expression levels and viral tropisms of HTLV-1 and HTLV-2, we reasoned that knowledge about the HTLV-3 receptors might provide insight into the tropism of this virus. We therefore generated vectors expressing HTLV-3 Env proteins and used them to begin to characterize the receptor complex used by HTLV-3 to bind and enter cells.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

In Vitro Analysis of Virus Particle Subpopulations in Candidate Live-Attenuated Influenza Vaccines Distinguishes Effective from Ineffective Vaccines

Two effective (vac⁺) and two ineffective (vac⁻) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac⁺ variants were distinguished from the vac⁻ variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac⁺) vaccines.Live-attenuated vaccines are considered more effective than their inactive or single-component counterparts because they activate both the innate and adaptive immune systems and elicit responses to a broader range of antigens for longer periods of time (2, 10, 25, 28). Influenza virus variants with alterations in the reading frame of the nonstructural NS1 protein gene (delNS1), which express truncated NS1 proteins, characteristically induce enhanced yields of type I interferon (IFN) relative to the yields of their isogenic parental virus encoding full-length NS1 proteins (11, 13, 21, 33, 39). Many of these delNS1 variants have proved to be effective as live-attenuated influenza vaccines (LAIVs), providing protection against challenge virus in a broad range of species (33, 46), including chickens (39, 44). The IFN-inducing capacity of the virus is considered an important element in the effectiveness of LAIVs (33). In that context, influenza viruses are intrinsically sensitive to the antiviral action of IFN (31, 32, 36), although they may display a nongenetic-based transient resistance (36). In addition, IFN sensitizes cells to the initiation of apoptosis by viruses (42) and by double-stranded RNA (40), which may be spontaneously released in the course of influenza virus replication (14). Furthermore, IFN functions as an adjuvant to boost the adaptive immune response in mammals (3, 4, 11, 26, 41, 43, 46) and in chickens when administered perorally in the drinking water of influenza virus-infected birds (19). This raises the question: does the enhanced induction of IFN by delNS1 variants suffice to render an infectious influenza virus preparation sufficiently attenuated to function as an effective live vaccine? To address that question, we turned to a recent report that described the selection of several variants of influenza virus with a common backbone of A/TK/OR/71-SEPRL (Southeast Poultry Research Laboratory) that contained NS1 protein genes which were unusual in the length and nature of the amino acid residues at the C termini of the truncated NS1 proteins that they expressed because of the natural introduction of a frameshift and stop codon by the deletion in the NS1 protein gene (44). delNS1 variants were isolated from serial low-inoculum passages of TK/OR/71-delNS1[1-124] (H7N3) in eggs (44). Four of these genetically stable plaque-purified variants, each encoding a truncated NS1 protein of a particular length, were tested as a candidate LAIV in 2-week-old chickens. Two of the delNS1 variants were effective as live vaccines (double deletions [D-del] pc3 and pc4) (phenotypically vac⁺), and two were not (D-del pc1 and pc2) (phenotypically vac⁻) (44), despite only subtle differences in their encoded delNS1 proteins. Why were they phenotypically different?The present study addresses this question by analyzing and comparing the different virus particles that constitute the subpopulations of these two effective (vac⁺) and two ineffective (vac⁻) live vaccine candidates. These analyses are based on recent reports in which noninfectious but biologically active particles (niBAPs) in subpopulations of influenza virus particles were defined and quantified (20, 21, 29). The study described in this report reveals several quantitative and qualitative differences between the particle subpopulations of the four candidate LAIVs, including the different types of IFN-induction dose-response curves, the quantum (maximum) yields (QY) of IFN induced, the efficacy of the interferon-inducing particles (IFPs), the replication efficiency of the virus, and the size of the plaques that they produced. Evidence is presented that the in vitro analysis of virus particle subpopulations may be useful to distinguish vac⁺ from vac⁻ LAIV candidates and provide a basis for identifying and enhancing the performance of particles with desirable phenotypes.     

Isolation of Basal Bodies with C-Ring Components from the Na+-Driven Flagellar Motor of Vibrio alginolyticus

To investigate the Na⁺-driven flagellar motor of Vibrio alginolyticus, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.The basal body, which is the part of the rotor, is composed of four rings and a rod that penetrates them. Three of these rings, the L, P, and MS rings, are embedded in the outer membrane, peptidoglycan layer and in the inner membrane, respectively (1), while the C-ring of Salmonella species is attached to the cytoplasmic side of the basal body (3). The C-ring is composed of the proteins FliG, FliM, and FliN (25), and genetic evidence indicates that the C-ring is important for flagellar assembly, torque generation, and regulation of rotational direction (33, 34). FliG, 26 molecules of which are incorporated into the motor, appears to be the protein that is most directly involved in torque generation (15). Mutational analysis suggests that electrostatic interactions between conserved charged residues in the C-terminal domain of FliG and the cytoplasmic domain of MotA are important in torque generation (14), although this may not be the case for the Na⁺-type motor of Vibrio alginolyticus (32, 35, 36). FliM interacts with the chemotactic signaling protein CheY in its phosphorylated form (CheY-P) to regulate rotational direction (30). It has been reported that 33 to 35 copies of FliM assemble into a ring structure (28, 29). FliN contributes mostly to forming the C-ring structure (37). The crystal structure of FliN revealed a hydrophobic patch formed by several well-conserved hydrophobic residues (2). Mutational analysis showed that this patch is important for flagellar assembly and rotational switching (23, 24). The association state of FliN in solution was studied by analytical ultracentrifugation, which provided clues to the higher-level organization of the protein. Thermotoga maritima FliN exists primarily as a dimer in solution, and T. maritima FliN and FliM together formed a stable FliM₁-FliN₄ complex (2). The spatial distribution of these proteins in the C-ring of Salmonella species was investigated using three-dimensional reconstitution analysis with electron microscopy (28). However, the correct positioning has still not been clarified.The Na⁺-driven motor requires two additional proteins, MotX and MotY, for torque generation (19-21, 22). These proteins form a unique ring structure, the T ring, located below the LP ring in the polar flagellum of V. alginolyticus (9, 26). It has been suggested that MotX interacts with MotY and PomB (11, 27). Unlike peritrichously flagellated Escherichia coli and Salmonella species, V. alginolyticus has two different flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum is used for motility in low-viscosity environments such as seawater (18). As described above, it is driven by a Na⁺-type motor. However, in high-viscosity environments, such as the mucus-coated surfaces of fish bodies, cells induce numerous unsheathed lateral flagella that have H⁺-driven motors (7, 8). We have been focusing on the Na⁺-driven polar flagellar motor, since there are certain advantages to studying its mechanism of torque generation over the H⁺-type motor: sodium motive force can be easily manipulated by controlling the Na⁺ concentration in the medium, and motor rotation can be specifically inhibited using phenamil (10). Moreover, its rotation rate is surprisingly high, up to 1,700 rps (compared to ∼200 rps and ∼300 rps for Salmonella species flagella and E. coli flagella, respectively) (12, 16, 17).Although understanding the C-ring structure and function is essential for clarifying the mechanism of motor rotation, there is no information about the C-ring of the polar flagellar motor of Vibrio species or the flagella of any genus other than Salmonella. Since Vibrio species have all of the genes coding for C-ring components, we would expect its location to be on the cytoplasmic side of the MS ring, as in Salmonella species. In this study, we attempted to isolate the polar flagellar basal body with the C-ring attached and investigate whether it is organized similarly to the H⁺-driven flagellar motor of Salmonella enterica serovar Typhimurium.